We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

BackgroundAGS-003 is an autologous immunotherapy prepared from fully matured and optimized monocyte-derived dendritic cells, which are co-electroporated with amplified tumor RNA plus synthetic CD40L RNA. AGS-003 was evaluated in combination with sunitinib in an open label phase 2 study in intermediate and poor risk, treatment naïve patients with metastatic clear cell renal cell carcinoma (mRCC).MethodsTwenty-one intermediate and poor risk patients were treated continuously with sunitinib (4 weeks on, 2 weeks off per 6 week cycle). After completion of the first cycle of sunitinib, patients were treated with AGS-003 every 3 weeks for 5 doses, then every 12 weeks until progression or end of study. The primary endpoint was to determine the complete response rate. Secondary endpoints included clinical benefit, safety, progression free survival (PFS) and overall survival (OS). Immunologic response was also monitored.ResultsThirteen patients (62%) experienced clinical benefit (9 partial responses, 4 with stable disease); however there were no complete responses in this group of intermediate and poor risk mRCC patients and enrollment was terminated early. Median PFS from registration was 11.2 months (95% CI 6.0, 19.4) and the median OS from registration was 30.2 months (95% CI 9.4, 57.1) for all patients. Seven (33%) patients survived for at least 4.5 years, while five (24%) survived for more than 5 years, including 2 patients who remain progression-free with durable responses for more than 5 years at the time of this report. AGS-003 was well tolerated with only mild injection-site reactions. The most common adverse events were related to expected toxicity from sunitinib therapy. In patients who had sequential samples available for immune monitoring, the magnitude of the increase in the absolute number of CD8+ CD28+ CD45RA− effector/memory T cells (CTLs) after 5 doses of AGS-003 relative to baseline, correlated with overall survival.ConclusionsAGS-003 in combination with sunitinib was well tolerated and yielded supportive immunologic responses coupled with extension of median and long-term survival in an unselected, intermediate and poor risk prognosis mRCC population.Clinical Trial Registry#NCT00678119

Immunogenicity, manufacturing feasibility, and safety of a novel, autologous dendritic cell (DC)-based immunotherapy (AGS-004) was evaluated in ten human immunodeficiency virus type 1 (HIV-1)-infected adults successfully treated with antiretroviral therapy (ART). Personalized AGS-004 was produced from autologous monocyte-derived DCs electroporated with RNA encoding CD40L and HIV antigens (Gag, Nef, Rev, Vpr) derived from each subjects's pre-ART plasma. Patients received monthly injections of AGS-004 in combination with ART. AGS-004 was produced within a mean of 6 weeks and yielded 4-12 doses/subject Full or partial HIV-specific proliferative immune responses occurred in 7 of 9 evaluable subjects. Responses were specific for the AGS-004 presented HIV antigens and preferentially targeted CD8 + cells. Mild adverse events included flulike symptoms, fatigue, and injection site reactions. No evidence of autoimmunity, changes in viral load, or significant changes in absolute CD4 + and CD8 + T cell counts were observed. This pilot study supports the further clinical investigation of AGS-004.

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-β. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-β, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

Saccharomyces cerevisiae strains expressing the activated RAS2vaI19 gene or lacking both cAMP phosphodiesterase genes, PDEI and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of a RAS2vaI9 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pdel-pde2-strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pdel-pde2-strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.The mammalian RAS genes were first discovered as homologs of retroviral oncogenes (1). Activated, mutant RAS alleles are frequently found in human tumors (2). RAS homologs have been found and described in many eukaryotic organisms (3)(4)(5)(6)(7). In the yeast Saccharomyces cerevisiae products ofthe two RAS homologs, RAS] and RAS2, activate adenylyl cyclase (8, 9). Although mammalian RAS proteins can function in this way when expressed in yeast (10,11), the function of mammalian RAS in mammalian cells is still unknown. One candidate target of mammalian RAS action is GAP, the GTPase-activating protein, which also has been proposed as a regulator of RAS action (12, 13).Like mammalian RAS, the yeast RAS2 gene can be activated by point mutation (14). Yeast containing RAS2v`l9 have multiple defects. They are sensitive to heat shock and cannot survive prolonged nutrient deprivation (8,15 (17). Yeast transformants were plated at 4103 colonies per plate on selective medium. Colonies were allowed to grow for 3 days and then were replica-plated onto preheated plates. Heat shocks were carried out at 550C for 10 min and followed by 2-3 days of recovery at 30'C. Surviving colonies were picked, restreaked on synthetic medium plates for colony purification, and then cultured in rich medium for 2-3 days to allow for plasmid loss from some cells. Of these cultures, 1 ,.d was plated onto YPD plates. After 2-3 days of growth, colonies were replica-plated onto synthetic medium plates (Leu+ selection), YPD plates, and YPD heat shock plates. Colonies were scored to ascertain if the observed heat shock resistance was plasmid dependent.Vector Construction and Cloning. The expression vector pADNS has been described (17). pADNS contains the alcohol dehydrogenase gene ADHI promoter immediately followed by unique HindIII and Not I cloning sites. pADANS was constructed as follows. A polymerase chain reaction (PCR) was carried out on the yeast ADHI gene in pJD14 (19). The first oligonucleotide primer (5'-TCTAAACCGTG-GAATATT) was placed within the promoter region of the gene and upstream of the EcoRV site within the promoter that is also contained in pADNS. The second primer (5'-GTCAAAGCTTCGTAGAAGAT...